(A) (Top Panel) Schematic representation of ER indicating domains conserved in the nuclear hormone receptor superfamily- activation function-1 (AF-1), DNA-binding domain (DBD), hinge region (Hinge) and Ligand-binding domain (LBD). (Bottom Panel) The conserved acetylation motif is highlighted in grey. The sequence methylated by SET7 in various known substrates is indicated. The asterisk indicates the target lysine residue.
(B) In vitro methylation assays were carried out using peptide substrates (H3 residues 1-24, ER residues 294-314) and three recombinant purified HMTases, SET7, DIM-5 and G9a (Collins et al., 2005; Zhang et al., 2003). Peptides (50 μM) were incubated with 0.1 μM HMTase at 37° C in the presence of ³H-labeled AdoMet. The reactions were terminated by adding SDS-PAGE loading buffer, resolved on a 20% polyacrylamide gel and analyzed by autoradiography.
(C) In vitro methylation assays were carried out using peptide substrates (ER: SPLMIKRSKKNSLALSLTADQ, GR: MNLEARKTKKKIKGIQQATTG, AR: MTLGARKLKKLGNLKLQEEGE, MR: MNLGARKSKKLGKLKGIHEEQ; ER-K303R: SPLMIKRSKRNSLALSLTADQ; ER-K303Ac: SPLMIKRSK(acetyl-K)NSLALSLTADQ) and recombinant SET7. Reactions contained 50 mM glycine pH 9.8, 1.8 μM [methyl-³H] AdoMet (14.9 Ci/mmol; NEN), 0.25 μM SET7 and 50 μM peptide and were incubated at 37 °C for 20 min. Methylation was analyzed either by precipitation with 20% TCA, collection on Whatman GF/F filters, and liquid scintillation counting (top) or by separation on 17% polyacrylamide-SDS gels and fluorography (bottom). Activity on the various peptides is calculated relative to wild-type ER Data represent the mean ± SD of two independent determinations.

(A) MCF7 and shSET7 cell lysates were analyzed by western blot using antibodies to ER, SET7 and GAPDH (loading control).
(B) (Left Panel) MCF7 and shSET7 cells were pulsed with methionine/cysteine-free media containing 100 μCi of L-³⁵S-methionine (Amersham Biosciences) for 1 hr followed by culture in regular growth media. Cells were harvested at various time points during the chase period and immunoprecipitated using an anti-ER antibody. Complexes were resolved by SDS-PAGE and the gel was fixed, dried and exposed to storage phosphor screens. A representative gel image is shown. Arrows indicate labeled ER. (Right Panel) The intensity of the labeled ER band was quantified by phosphorimage analysis using ImageQuant software (Molecular Dynamics) and the percent ER remaining was calculated relative to that at the beginning of the chase period (time zero). The mean values from two independent experiments are shown.
(C-F) MCF7 cells (C, D) or shSET7 cells (E, F) were transfected with expression constructs containing Flag-tagged wildtype (WT) or mutant (K303A, K303R) ER. After 1 day, cells were labeled in L-³⁵S-methionine and subjected to pulse chase analysis as described in A except that immunoprecipitations were carried out using Flag-M2 agarose. (C, E) Representative gel images are shown. (D, F) The intensity of the labeled ER was quantified by phosphorimage analysis using ImageQuant software (Molecular Dynamics) and the percent ER remaining calculated relative to time zero. The mean values from two independent experiments are shown.

(A) MCF7 and shSET7 cells were cultured in EFM for 3 days and then treated with vehicle alone (-) or 100 nM E₂ (+) for 20 min. Samples were split and whole cell extracts (WCE) were prepared by lysis in RIPA buffer. Nuclei were prepared from the remaining cells and extracted with 50mM Tris-HCl, 0.42M KCl, 1mM MgCl₂, 10%glycerol, 1mM DTT, 0.1mM PMSF to generate soluble nuclear extracts (NE). Equal cell equivalents were fractionated by SDS-PAGE and subject to western blotting using antibodies to ER, SET7, and DNA polymerase δ (loading control).
(B) Estrogen-induced recruitment of ER to the PS2 and PgR genes was analyzed by ChIP. MCF7 (filled diamonds) and shSET7 cells (open triangles) were cultured in EFM for 3-4 days and then treated with 100 nM E₂. At the indicated time points, ChIP was performed with antibodies directed against ER. Immunoprecipitated DNA was analyzed by real-time PCR using primers encompassing the EREs of the PS2 and PgR promoters and a putative PgR enhancer #1 (Carroll, et al., 2006). The E₂-stimulated fold increase in recruitment was determined relative to time zero after normalization to input DNA. The mean of three (MCF7) or two (shSET7) independent experiments analyzed in triplicate is shown.
(C-D) MCF7 cells were grown in EFM and transfected with pERE-Luc, pRL-Tk and pMyc-SET7. After 24 hr, the cells were treated with vehicle alone (-) or 1 pM E₂ (+). (C) Luciferase activity was determined relative to the unstimulated cells after normalization to the Renilla luciferase internal control. The mean ± SD from two independent experiments performed in triplicate is shown. (D) Protein lysates used in the luciferase assay were immunoblotted with the indicated antibodies. SET7 appears as a doublet; the upper band is Myc-tagged SET7 and the lower band is endogenous SET7.

(Upper) Methylation of ER at K302 by SET7 could prevent the ubiquitylation of K302 by an E3 ubiquitin ligase, such as the BRCA1/BARD1 complex. Loss of SET7-mediated methylation at K302 would allow ubiquitylation of K302, and premature proteasome-mediated degradation of ER. (Lower) Methylated ER may recruit specific proteins, such as CaM or a methyl lysine binding protein. These methyl-K302-specific binding proteins would then directly or indirectly block the action of E3 ligases and ER degradation. Subsequently, these binding proteins may be released to allow the ubiquitylation and degradation of methylated ER after it completes the transactivation cycle.

(A) HeLa cells were transfected with expression constructs for Myc-SET7 and wild-type (WT) or mutant (K302R, K303R)) ER, or a vector control (-). (Top) After 48 hr, ER was immunoprecipitated from 1 mg protein lysate using an anti-ER antibody and analyzed by western blotting with anti-ER K302me1 or anti-ER antibodies. (Bottom) One-tenth of input was analyzed by western blotting with the indicated antibodies.
(B) MCF7 cells were transfected with expression constructs for Myc-SET7 and wild-type (WT) or mutant (K302R) ER or vector (-). (Upper left) After 48 hr, total ER was immunoprecipitated from 1 mg protein lysates using an anti-ER antibody and analyzed by western blot with the anti-ER K302me1 or anti-ER antibodies. (Upper right) The same protein lysates (1 mg) used in lanes 5 and 7 were immunoprecipitated with the anti-ER K302me1 antibody and analyzed by western blot with an anti-ER antibody. (Bottom) One-tenth of input was analyzed by western blot with the indicated antibodies.

(A) MCF7 and shSET7 cells were grown in EFM for 4 days and transfected with pERE-Luc, pRL-Tk and pNCoA2. One day later, the cells were treated with either ethanol vehicle alone (−) or 1 pM E₂ (+) for 24 hr. The fold-increase in firefly luciferase activity compared to that of unstimulated cells in the absence of NCoA2 was determined, after normalization to Renilla luciferase activity. Shown is the mean ± SD of two independent experiments performed in triplicate.
(B-C) MCF7 and its derivatives were cultured in EFM for 4 days, followed by treatment with ethanol vehicle (V) or 1 nM E₂ for 1, 2 or 24 hr after which RNA was isolated, reverse-transcribed and analyzed by real-time PCR using primers specific for PS2, PgR, or 18s rRNA. The E₂-stimulated fold-increase in PS2 (B) and PgR (C) mRNA levels were determined relative to time zero, after normalization to 18s rRNA. Shown is the mean ± SD of two independent experiments performed in triplicate.

(A) The reaction occurred during crystallization, the methyl group has transferred to the ε-amino of ER-K302 and AdoMet is converted to AdoHcy, which is still present in the complex (right panel). The AdoMet analog sinefungin (adenosyl ornithine) was used to prepare a ternary complex mimicking the step prior to methyl transfer because it also carries a formal positive charge on the ε amino group (middle panel). The substrate peptide and sinefungin, or the methlyated peptide and the reaction product AdoHcy, are located at the opposite ends of the target lysine-binding channel. The atoms are colored red, nitrogen; blue, oxygen; grey, carbon; and yellow, sulfur.
(B) Three pairs of salt bridges and hydrogen bonds define SET7-ER-peptide interactions (dashed lines). Inset is a surface representation of the peptide-binding groove.